Borrelia burgdorferi (Bb), the causative agent of Lyme disease, is maintained in a complex enzootic cycle involving its arthropod vector (Ixodes ticks) and a rodent mammalian host. The key strategy that Bb has evolved to adapt to two markedly different host environments is the dramatic alteration of its surface proteins during the process of tick feeding. In the past few years, we and others have uncovered a novel regulatory pathway, the Rrp2-RpoN-RpoS pathway (Rrp2 pathway), as a major mechanism underlying differential gene expression during the spirochete's enzootic cycle. The discovery of the Rrp2 pathway engenders two fundamental questions that are critical to our understanding of host adaptation by Bb. (i) What are the virulence factors controlled by the Rrp2 pathway? (ii) What are the upstream signaling events that activate the Rrp2 pathway? Our long-term objective is to elucidate how Bb causes disease by identifying Bb virulence factors and deciphering the mechanisms underlying their regulation. To accomplish this objective, we have formulated the following three specific aims: i) To characterize candidate virulence factors controlled by the Rrp2 pathway. ii) To determine the role of Hk2 in Rrp2 activation during tick transmission and mammalian infection. We will test the hypothesis that Hk2 is important to Rrp2 activation in vivo and to the infectious cycle of Bb. iii) To better define the signaling sensing mechanisms for the Hk2-dependent and Hk2-independent pathways for activation of Rrp2 in vivo and in vitro. We will test the hypothesis that the PAS domain of Hk2 is the receptor for mammalian host-specific signals, whereas acetyl-P is one of the factors that contribute to the Hk2-independent Rrp2 activation in vitro and in vivo. PUBLIC HEALTH RELEVACE Accomplishing Specific Aim 1 will identify new virulence factors that are important to mammalian infection. Outcomes of Aims 2 and 3 will elucidate the complex signal sensing mechanisms of the Rrp2 pathway, which will fill a major gap in our understanding of Bb host adaptation. These findings could lead to the developments of therapeutic targets and form a basis for developing strategies to block the enzootic cycle of Bb. The proposed work will also allow us to gain insight into host adaptation of other vector-borne bacterial pathogens.